Abstract

Background

In an effort to develop novel treatments for communicating hydrocephalus, we have shown previously that the transforming growth factor-β antagonist, decorin, inhibits subarachnoid fibrosis mediated ventriculomegaly; however decorin’s ability to prevent cerebral cytopathology in communicating hydrocephalus has not been fully examined. Furthermore, the capacity for diffusion tensor imaging to act as a proxy measure of cerebral pathology in multiple sclerosis and spinal cord injury has recently been demonstrated. However, the use of diffusion tensor imaging to investigate cytopathological changes in communicating hydrocephalus is yet to occur. Hence, this study aimed to determine whether decorin treatment influences alterations in diffusion tensor imaging parameters and cytopathology in experimental communicating hydrocephalus. Moreover, the study also explored whether diffusion tensor imaging parameters correlate with cellular pathology in communicating hydrocephalus.

Methods

Accordingly, communicating hydrocephalus was induced by injecting kaolin into the basal cisterns in 3-week old rats followed immediately by 14 days of continuous intraventricular delivery of either human recombinant decorin (n = 5) or vehicle (n = 6). Four rats remained as intact controls and a further four rats served as kaolin only controls. At 14-days post-kaolin, just prior to sacrifice, routine magnetic resonance imaging and magnetic resonance diffusion tensor imaging was conducted and the mean diffusivity, fractional anisotropy, radial and axial diffusivity of seven cerebral regions were assessed by voxel-based analysis in the corpus callosum, periventricular white matter, caudal internal capsule, CA1 hippocampus, and outer and inner parietal cortex. Myelin integrity, gliosis and aquaporin-4 levels were evaluated by post-mortem immunohistochemistry in the CA3 hippocampus and in the caudal brain of the same cerebral structures analysed by diffusion tensor imaging.

Electronic supplementary material

Background

Hydrocephalus is a common paediatric neurosurgical presentation with an incidence of 0.48–0.81 per 1000 live births [1, 2, 3]. Communicating hydrocephalus is aetiologically heterogeneous; bacterial meningitis, subarachnoid haemorrhage, trauma, intracranial and intraspinal tumours as well as leptomeningeal metastases can all cause the disorder [4, 5, 6, 7, 8, 9, 10]. The incidence of communicating hydrocephalus following subarachnoid haemorrhage is at least 13 % and can be as high as 67 % [11]. In addition to ventriculomegaly, communicating hydrocephalus is accompanied by extensive global cerebral pathology, including widespread reactive gliosis, hydrocephalic oedema and demyelination [10, 12].

Although shunting is the current standard of care for children with hydrocephalus, the procedure is associated with severe complications that contribute to an increased patient morbidity [13, 14, 15, 16]. Furthermore, academic attainment and social integration difficulties continue into adulthood for those with the disease [17, 18, 19]. Therefore, the development of novel therapeutic strategies to prevent the development of hydrocephalus or promote recovery is of critical importance. Our recent study (Additional file 1: Figure S1) supports the key role of transforming growth factor-beta (TGF-β) in communicating hydrocephalus, as decorin, a TGF-β antagonist [20, 21, 22, 23] ameliorated subarachnoid fibrosis and therefore significantly attenuated the enlargement of the ventricular system [12]. However, the effectiveness of decorin to prevent cytopathology in hydrocephalus is yet to be examined thoroughly. Given that cellular pathology is largely responsible for the array of functional deficits observed clinically and contributes to the impairment in patient health-related quality of life, it is important to understand whether decorin can attenuate these alterations in vivo [10, 24, 25].

Greater insight into the cytopathological changes occurring in communicating hydrocephalus can be achieved with the use of advanced non-invasive magnetic resonance diffusion tensor imaging (DTI) [26]. DTI is a specialised magnetic resonance imaging (MRI) technique that examines tissue anisotropic properties and cerebral microstructural integrity [27, 28]. DTI yields a set of quantitative metrics, reflecting the magnitude along the principal axes of water diffusion, which are sensitive to changes in the underlying brain microstructure. Commonly used scalar DTI parameters such as axial (AD), radial (RD), and mean diffusivities (MD) (equivalent to the speed of motion in the principal axes of diffusion) or the fractional anisotropy (FA) (equivalent to a normalized aspect ratio of the principal axes of diffusion) have been useful in the investigation of cerebral abnormalities; an increase in the AD, RD and MD alongside a decrease in the FA occurs in the cerebral white matter of children with hydrocephalus [29, 30, 31, 32, 33]. Furthermore, the specificity of DTI to act as a surrogate measure of cerebral pathology has been highlighted in a variety of conditions, including hypoxic ischaemic injury [34, 35], multiple sclerosis [36, 37, 38, 39], spinal cord injury [40], obstructive hydrocephalus [41], temporal lobe epilepsy [42, 43] and for delineating gliomas [44]. However, correlations between DTI parameters and underlying cytopathology in communicating hydrocephalus have yet to be determined (Appendix 1).

Therefore, using immunohistochemistry and clinically relevant neuroimaging we investigated whether decorin is able to attenuate damage-related parameters and if cellular changes in communicating hydrocephalus can be quantitatively characterised by DTI using a juvenile rat model of the disorder.

Methods

Experimental animals

Three-week-old Sprague–Dawley rats (Charles River, Massachusetts, USA) were housed in litters in individual cages, kept under a 12 h light/dark cycle with free access to food and water. Animals were monitored for adverse effects of treatments, such as distress, lethargy, weight loss and seizures, and any animals showing severe adverse effects were euthanised. Experiments were conducted at the University of Utah in accordance with the guidelines of the National Institutes of Health Care and Use of Laboratory Animals and approved by the University of Utah Ethics Committee.

Experimental design and surgical techniques

The experimental design and surgical techniques are described in detail elsewhere [12]. Using a ventral approach, the interval between the occipital bone and the C-1 vertebral body was exposed and a 30 gauge angled needle was inserted into the prepontine (basal cistern) subarachnoid space. 30 µl of 20 % kaolin solution (200 mg/ml in 0.9 % sterile saline; Fisher Scientific, Massachusetts, USA) was injected to induce communicating hydrocephalus and the rat was either allowed to recover or underwent osmotic pump and intraventricular cannula implantations. The cannulae were inserted into the right lateral ventricle and fixed in place with glue and bone cement (Biomet UK Ltd, Bridgend, UK) to a stabilising screw, and connected to subcutaneously implanted mini osmotic pumps. Osmotic pumps (model 2002 adapted for use in MRI scanners with PEEK tubing, Alzet, Durect Corporation, California, USA) were filled with either 5 mg/ml human recombinant decorin (GalacorinTM, Catalent/Pharma Solutions, New Jersey, USA) or 10 mM phosphate buffered saline (PBS) pH 7.4 (Sigma-Aldrich, Missouri, USA). Over the subsequent 14 days, human recombinant decorin was infused at a rate of 2.5 mg/0.5 ml/h.

Magnetic resonance imaging and diffusion tensor imaging

Imaging experiments were conducted 14 days post injury using a 7-Tesla horizontal-bore Bruker Biospec MRI scanner (Bruker Biospin, Ettlingen, MA, USA) interfaced with a 12-cm actively shielded gradient insert capable of producing magnetic field gradient up to 600 mT/m. Animals were anesthetised using 1–3 % Isoflurane and 0.8 L/min O2 and their vital signs (respiration, temperature, heart rate and oxygen saturation percentage) were continuously monitored using a MR-compatible physiological monitoring system (SA Instruments, Stony Brook, NY, USA). Animals were placed in a 72-mm volume coil for signal transmission, and a quadrature surface coil was placed on the head for signal reception. Acquisition of T2-weighted MRI scans and ventricular volume analysis has been described previously [12]. DTI scans were conducted using spin echo diffusion-weighted sequences with single-shot EPI readout, with the following parameters (TR of 3760 ms, TE of 44 ms, 15 coronal 1 mm-thick slices, a field of view of 2.5 × 2.5 cm, and an in-plane resolution of 195 × 195 µm). Thirty uniformly-spaced over unit sphere diffusion-weighted gradient directions and five non-weighted images were acquired with two signal averages and the following diffusion parameters: diffusion gradient duration 7 ms, separation 20 ms, diffusion encoding sensitivity 700 s/mm2. Scan time was 4 min. For ventriculomegaly analysis, one MRI scan image was chosen from the rostral cerebrum (−0.36 mm from bregma) and the caudal cerebrum (−3.72 mm from bregma) for each rat, and the ventricular area was determined in each scan using ImageJ.

DTI voxel based analysis

Prior to commencing voxel-based analysis, double blinding was introduced to prevent group identification. Using the software, DSI Studio (DSI Studio, Pittsburgh, PA), DTI images were reconstructed and processed to produce voxel based maps, from which regions of interest (ROIs) could be analysed. The seven ROIs selected include: corpus callosum, periventricular white matter, caudal and rostral internal capsule, outer parietal cortex, inner parietal cortex and CA1 hippocampus. DTI parameter values from four serial sections (1.28 mm anterior to Bregma to 3.72 mm posterior to Bregma) of the corpus callosum and periventricular white matter were analysed in a total of 17 rats [Intact (n = 4), kaolin (n = 4), kaolin + PBS (n = 5), kaolin + decorin (n = 4)]. The rostral corpus callosum and periventricular white matter sections were defined as 1.28 and −0.36 mm from Bregma. The caudal corpus callosum and periventricular white matter sections were derived from −2.76 to −3.72 mm from Bregma (Fig. 2a). The remaining five ROIs were analysed in 16 rats, with four animals being examined in each experimental group. Three sections were independently analysed for the CA1, caudal internal capsule, outer and inner parietal cortex from 0.36 to 3.72 mm posterior to Bregma. An average of two sections from 1.28 mm anterior to Bregma to 0.36 mm posterior to Bregma were individually analysed for the rostral internal capsule. ROIs were identified using a FA voxel based map and an analogous DTI image (Fig. 1). The mean FA, MD, AD and RD values were calculated for each ROI of each animal.

Pixel based analysis of immunofluorescent staining

Quantitative analysis was undertaken using the software, Image J and all analyses were undertaken with the operator masked to the experimental group. Images for each immunofluorescent stain were processed identically before being analysed. For each image analysed, four randomly placed regions of interest (ROIs) were drawn with each ROI being 2.96 mm wide and 1.57 mm in height. For the corpus callosum, periventricular white matter and CA1 and CA3 hippocampal regions, a mean of 16 ROIs (four regions of interest × four coronal sections) were chosen per rat per stain. An average of 8 ROIs (four regions × two coronal sections) were selected for the internal capsule, caudate-putamen, parietal cortex and occipital cortex. All areas were analysed for GFAP, OX-42 and AQP4 staining however, as MBP is a marker of white matter integrity, only the corpus callosum, periventricular white matter and internal capsule were analysed for this antibody.

GFAP and OX-42 image processing included the conversion of images into a gray scale format prior to spatial filtering, thresholding and despeckling of the images using Image J. Images stained for AQP4 and MBP were identically processed to the GFAP and OX-42 images except thresholding was not performed. The mean percentage area of GFAP, OX-42, AQP4 and MBP positive staining, for each experimental group was calculated.

Bright field microscopy

In order to assess hippocampal size, one cerebral section, at least 2.5 mm posterior to Bregma from each experimental animal was examined at ×10 magnification using the Nikon SM21500 dissecting microscope (Nikon, Tokyo, Japan). Images were captured with a Nikon ds-2mv high-resolution camera (Nikon). Hippocampal area was assessed by using the Image J software analyze area tool.

Statistics

Statistical analysis was conducted using SPSS software, version 22 (IBM, Armonk, NY). Normally distributed data were analysed using a one-way ANOVA followed by a post hoc Tukey test. In the absence of normality, data were analysed using the Kruskal–Wallis test and tested for significant pairwise comparisons. Normally distributed data were expressed as the mean ± standard error of the mean (SEM). Correlation analysis was performed using a two-tailed Spearman’s correlation test. As immunohistochemistry analysis was performed on caudal sections, mean DTI data from Section 2.76 and 3.72 mm posterior to Bregma were used for correlation analysis. Correlation analysis was not undertaken for the inner parietal cortex, caudate-putamen, occipital cortex or CA3 region of the hippocampus because DTI analysis was not being performed in these areas. Values were considered statistically significant when p values were *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.

Significant changes in the mean differences between the rostral versus caudal ventricular volumes amongst the four experimental groups

p

Intact vs Kaolin

0.005

Intact vs Kaolin + PBS

<0.001

Intact vs Kaolin + decorin

0.946

Kaolin vs Kaolin + PBS

0.530

Kaolin vs Kaolin + decorin

0.015

Kaolin + PBS vs Kaolin + decorin

0.001

Rostrally, ventricular volume significantly correlated with the FA, MD, AD and RD measurements of the corpus callosum and periventricular white matter (Table 2). Likewise, the caudal ventricular volume correlated with all DTI parameter measures in the corpus callosum and all except the AD in the periventricular white matter (Table 2).

No significant differences in AQP4, OX-42 and GFAP immunostaining were present between the experimental groups in the caudate putamen, parietal cortex and occipital cortex (Additional file 4: Table S2). However, in the corpus callosum, periventricular white matter, caudate-putamen, parietal cortex and occipital cortex, GFAP immunostaining positively correlated with AQP4 levels (Table 3). No significant correlations were present between OX-42 and AQP4 levels in any of the regions of interest.

Table 3

AQP4 levels correlated significantly with the marker of gliosis, GFAP, in the corpus callosum, periventricular white matter, caudate-putamen and parietal and occipital cortex

Region of interest

R

p

Corpus callosum

0.614

0.005

Periventricular white matter

0.854

<0.001

CA1 hippocampus

0.332

0.166

CA3 hippocampus

0.446

0.056

Internal capsule

0.291

0.226

Caudate-putamen

0.495

0.043

Parietal cortex

0.528

0.020

Occipital cortex

0.607

0.006

Statistically significant correlations = p < 0.05

R correlation coefficient

Hydrocephalic cytopathology correlates with abnormalities on DTI

In the caudal corpus callosum, increased astrocyte (GFAP) and AQP4 levels positively correlated with the AD (Table 4). Furthermore, in the caudal periventricular white matter (Table 4), GFAP and AQP4 positively correlated with AD, MD and RD. Moreover, the presence of cytopathology discouraged anisotropic water diffusion in the caudal periventricular white matter as the FA negatively correlated with astrocyte (GFAP), microglial (OX-42) and AQP4 immunostaining. A negative correlation was also present between myelin levels (MBP) and the MD of the caudal periventricular white matter.

Table 4

The marker of gliosis, GFAP, and AQP4 levels correlated with DTI parameter values in the periventricular white matter

Discussion

This study demonstrates that decorin is able to protect and maintain DTI parameter values at normality in the caudal corpus callosum and caudal periventricular white matter. Likewise, decorin prevents astrogliosis and oedema in the caudal periventricular white matter and preserves myelin integrity in the caudal internal capsule. Furthermore, cytopathology in communicating hydrocephalus is predominantly localised to the caudal cerebrum. Moreover, DTI parameters correlate with cytopathology specifically in the caudal periventricular white matter. DTI is therefore a useful tool to act as a surrogate measure of cytopathology in communicating hydrocephalus.

Recent studies in post-haemorrhagic hydrocephalus suggest that occipital horn enlargement is greater and precedes the dilation of the remaining ventricular system [46, 47, 48]. This asymmetry is a pattern that is repeated in other types of hydrocephalus including congenital hydrocephalus [49] and idiopathic chronic hydrocephalus [50], although this feature has not been explored in depth or quantitatively. In feline infants [51], neonatal rats [41, 52] and adult dogs [53] with non-communicating hydrocephalus induced by kaolin injections into the cisterna magna, the occipital horns of the lateral ventricles are conspicuously larger than the frontal horns. Our results support these findings, albeit in an experimental model of communicating hydrocephalus, by showing that caudal portions of the lateral ventricles expand more than frontal regions, and DTI abnormalities are largely situated in the caudal white matter.

Asaaf and colleagues [54] suggested that DTI could be used as a marker of white tissue compression in obstructive hydrocephalus. Furthermore there have been no observed DTI changes in the white matter of idiopathic intracranial hypertension patients (high ICP but no ventriculomegaly [55]) suggesting that compression of tissue impacts DTI parameters. In the caudal periventricular white matter, our findings replicate the abnormalities in the MD, AD and RD observed in hydrocephalic children [29, 30, 31]. In contrast to our findings, the MD of the periventricular white matter does not increase in post-haemorrhagic hydrocephalus in adults [56], therefore the maturity of the brain appears to influence DTI alterations. Similar to the findings of Yuan et al. [41] in rats of the same age with obstructive hydrocephalus (blockage of the cisterna magna), our communicating hydrocephalic animals display an increase in MD and reduced FA in the caudal corpus callosum. Our study has also revealed an increase in the AD and RD of the caudal corpus callosum in communicating hydrocephalus, which is additionally preventable by decorin treatment.

The cytopathology observed in our model supports current literature and is largely preventable with decorin treatment [10, 12, 57, 58, 59, 60, 61, 62, 63, 64]. Although white matter abnormalities discovered were similar to those in hydrocephalic children [29, 30, 31, 32, 33, 34], decorin was only able to protect the internal capsule from myelin damage. TGF-β mediated signaling promotes central nervous system myelination by enhancing oligodendrocyte progenitor cell differentiation and maturation [65, 66]. It is possible that internal capsule oligodendrocyte progenitor cells may be more susceptible to abnormalities in TGF-β signaling than those of the corpus callosum or periventricular white matter, hence explaining the observed result.

The relationship between DTI parameters and cerebral histopathological changes has been discussed extensively in recent literature [27, 31, 32, 67, 68, 69, 70, 71]. Events that discourage directional water movement, such as interstitial oedema and neurodegeneration cause a decline in the FA [27, 28, 72, 73, 74, 75, 76, 77]. The AD and RD are two DTI parameters that influence the FA and provide insight into axonal and myelin integrity, respectively [73, 76]. Both the AD and RD are also influenced by gliotic tissue changes [27, 72, 76]. Furthermore, an increase in average amount of diffusion in a given volume of tissue, caused by the presence of interstitial oedema or the loss of cellular barriers, results in a rise in the MD [27, 77, 78].

Consistent with the results of Yuan et al. [41] in juvenile rats with obstructive hydrocephalus, our findings in communicating hydrocephalus show positive correlations between GFAP increases and MD, AD, and RD in caudal periventricular white matter. Likewise, the increased levels of OX-42 (a marker of microglia) correlated negatively with the FA. This result may seem surprising since cytoarchitecturally in the periventricular white matter of kaolin and kaolin + PBS rats, the majority of microglial processes were longitudinally oriented; therefore an increase in FA would have been predicted [58]. However, as others have reported in congenital hydrocephalus [64], the cell bodies of reactive microglia in the periventricular white matter of our kaolin and kaolin + PBS animals were enlarged and widened. This cytopathological characteristic may have obstructed the parallel diffusion of water causing the FA to decrease. Since the pathophysiology of hydrocephalus is extremely multifactorial, it is unlikely that glial alterations alone exert a causative effect on DTI parameters.

The MBP levels of the caudal periventricular white matter correlate with the MD. These results corroborate the current literature; by increasing the volume of the extracellular space, myelin disorganisation and demyelination increases the MD of water molecules [27, 73, 76]. In support of the Tourdias et al. [78] report on communicating hydrocephalus, AQP4 levels positively correlated with the MD measurements in the caudal periventricular white matter. AQP4 levels also positively correlated with the FA, RD and more interestingly the AD measurements. Although sparse literature exists on the relationship between AQP4 and AD, we suggest that the removal of excess interstitial fluid by high levels of AQP4 may promote the unobstructed parallel movement of water through the periventricular white matter, hence resulting in an increase in the AD measurement. Further investigation of this hypothesis needs to be undertaken in order to substantiate such claim.

Here we have used kaolin to induce communicating hydrocephalus to help us determine the therapeutic effects of decorin. It is important to recognize the possibility that some decorin-treated animals may not have developed ventriculomegaly simply because of induction failures. However, it is unlikely that a significant proportion of the decorin-treatment group would not develop ventriculomegaly given the fact that 82 % of kaolin-only or kaolin + PBS animals demonstrated significantly enlarged ventricles [12]. In addition, 79 % of adult rats with identical induction procedures developed ventriculomegaly [79]. Thus, we believe that the improvements in the decorin-treated animals were due primarily to the drug intervention. Another consideration of the study is that the kaolin model of hydrocephalus is not the most clinically relevant model, however it is the best characterised and most widely used, successfully replicating the development and pathophysiological consequence of acquired hydrocephalus. Kaolin induces an inflammatory response with concomitant deposition of fibrosis in areas of the subarachnoid space close to the injection site [80, 81] which is very similar to that observed in subarachnoid haemorrhage rat models [82]. The next step would be to determine the effects of decorin in a post haemorrhagic model. Recently, Yan et al. [83] demonstrated that pretreating rats with decorin in a subarachnoid haemorrhage model led to a reduction in ventriculomegaly and markers of fibrosis, indicating that decorin may have beneficial effects in subarachnoid haemorrhage. However further work needs to be conducted looking at the changes in cerebral cytopathology and microstructure with decorin treatment in this model.

Conclusions

Our findings highlight the therapeutic potential of decorin to attenuate hydrocephalus-induced changes in astrogliosis, oedema and demyelination, particularly in the caudal periventricular white matter. Our study also helps to validate the use of DTI as a surrogate marker of cytopathology in communicating hydrocephalus and demonstrates that the caudal region of the brain appears to be the most affected, showing the greatest changes in ventriculomegaly, DTI and cytopathological measures in our experimental model.

Acknowledgements

Anuriti Aojula was funded by the Sir Arthur Thompson Trust. Dr. Hannah Botfield was funded by the BBSRC. Professor James Patterson McAllister II was funded by the Department of Neurosurgery at the University of Utah, the Pediatric Hydrocephalus Association, the University of Utah Vice President for Research and the Department of Neurosurgery, Washington University School of Medicine. Dr. Alexandra Sinclair is funded by an NIHR Clinician Scientist Fellowship (NIHR-CS-011-028) and by the Medical Research Council, UK (MR/K015184/1).

Competing interests

The authors declare that they have no competing interests.

Appendix 1

Diffusion tensor imaging (DTI). DTI is a specialised magnetic resonance imaging technique that is used to gain greater appreciation of white matter disease-related pathophysiology via probing the random translational motion of water molecules [26]. The scalar parameters in DTI provide a quantitative method to assess cerebral water motion by specifically examining the magnitude and direction of water diffusion, which is quantified by measuring key parameters such as axial diffusivity (AD), radial diffusivity (RD) and their derivatives mean diffusivity (MD) or fractional anisotropy (FA). The FA values provide insight into the anisotropy of water diffusion. Water may diffuse isotropically, i.e. equally in all directions, or along a specific direction, therefore becoming anisotropic in nature. Moreover, the FA can be influenced by changes in microstructural integrity [27]; neurodegeneration and axonal reorganization hinders isotropic water movement, decreasing the FA. Furthermore, in ventriculomegaly-induced cerebral compression, increased AQP4 levels and gliosis raise the FA [74]. The RD and AD are two parameters that directly influence the FA. In white matter, the proportion of water diffusing perpendicular to neuronal fibres is assessed by RD, whilst the degree of water diffusion parallel to tract orientation is determined by the AD. Increased RD and AD are indicators of myelin and axonal integrity, respectively. Both RD and AD are also reported to increase upon astrogliosis [27]. Quantification of the average magnitude of diffusion in a given volume of tissue is provided by the MD value. MD is decreased by the presence of cellular barriers [80]. In contrast, interstitial edema and greater AQP4 and microglial presence are responsible for a rise in the MD [80].

Additional file 3: Table S1. In the corpus callosum, no significant cytopathological changes were observed in hydrocephalic animals. The mean values ± the standard error of the means of GFAP, OX-42, AQP4 and MBP immunostaining in the four different experimental groups are expressed.

Harris C, McAllister JP. What we should know about the cellular and tissue response causing catheter obstruction in the treatment of hydrocephalus. Neurosurgery. 2012;70:1589–601 (discussion 1601–1602).CrossRefPubMedGoogle Scholar

Copyright information

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.